PROJECT SUMMARY Flow cytometry for identification of subpopulations and microscopy for cellular imaging are critical tools for analyzing a broad range of tissues and processes, and have become staple technologies for modern biological and biomedical investigations. However, it remains challenging to combine these techniques and perform imaging on rare cell populations in a high-throughput manner. To address those limitations, the Amnis ImageStream-X Mark II imaging flow cytometer has been developed by MilliporeSigma. This unique technology is capable of generating both traditional flow cytometric fluorescence intensity measurements as well as imaging data for each cell or particle analyzed. This unique combination allows visualization of subcellular location of fluorescence with the high-throughput, speed, and sensitivity of flow cytometry, enabling experiments that would be very technically challenging otherwise, if not impossible, using either application alone. This including cell signaling, cell cycle and mitosis, internalization and co-localization, surface vs. intracellular co-localization, DNA damage & repair, stem cell biology, microbiology, cell-cell interaction, shape change and chemotaxis, immunological synapse, micronucleus counting, just to name a few. Currently, this cutting-edge technology is not readily available to the Columbia University Irving Medical Center (CUIMC) research community. There are currently three other ImageStream instruments in the New York City area, but the location and/or capabilities of each of these instruments preclude them from being a realistic and useful solution to the needs of Columbia University researchers. Based on a prospective survey of CUIMC scientists, we have identified at least 8 NIH-funded major users as well as a large number of minor and other users. The proposed applications are diverse, leveraging the various scientific strengths of a number of CUIMC laboratories. Some examples include Dr. Emmanuelle Passegu's work on hematopoietic stem and progenitor cells and emergency myeloid regeneration pathways, Dr. Nadeen Chahine's ongoing studies of inflammation on the cellular biomechanics and mechanotransduction properties of intervertebral spinal discs, and Dr. Jordan Orange's investigations of human natural killer cells in controlling viral infections and malignancy development. The leadership of CUIMC, the Columbia Stem Cell Initiative, directed by Dr. Passegu, the Department of Pediatrics, chaired by Dr. Orange and the Department of Orthopedics Surgery are strongly supportive of the acquisition of this instrument, and will provide additional funds beyond those requested in the S10 application to help manage its operation. In addition, dedicated space has been allocated within the newly-established CSCI Flow Cytometry Core facility where the instrument will be located and managed. We predict that the establishment of an Amnis ImageStream-X Mark II imaging flow cytometer on the CUIMC campus will be pivotal in driving new scientific insights across a number of NIH-funded projects and biomedical disciplines.